Hello David:

My lab does histology on fish ranging from about 15mm on up.

I will ask first: why not split the fish anterior-posterior and place the
head in 95% ethanol and the body posterior/ventral of the otoliths in your
fixative of choice for gonads (I'd use 10% NBF over Bouin's any day)?  Are
you sectioning the otoliths or polishing them for age studies?  In other
words, are you removing the otoliths prior to dehydration and embedding?  If
so, why the need to keep the fish intact in the first place?

If you can't bisect the fish, in my experience sodium phosphate buffered NBF
does not chelate calcium.  We breadloaf Atlantic menhaden in the 145mm range
and fix in 10% NBF.  These fish are quite bony (they are herrings) and must
be decalcified before processing and sectioning.

I hope this helps.

Regards -
Carol
*********************
Carol B. McCollough, HT/HTL(ASCP)
Diagnostics & Histology Laboratory Manager
Maryland Department of Natural Resources
Cooperative Oxford Laboratory
904 S. Morris Street
Oxford, Maryland 21654
cmccollough@dnr.state.md.us


-----Original Message-----
From: Dave Allsop [mailto:David.Allsop@ed.ac.uk]
Sent: Wednesday, January 30, 2002 10:59 PM
To: histonet@pathology.swmed.edu
Subject: Optimising gonad preservation whilst retaining otolith
microstructure


Dear Histonet

I am collecting populations of very small fish from remote locations, and
need to extract two important pieces of information from them for my study.
First I need to access the cellular morphology of the gonad for sex and
maturity estimates, and second I need to access the age and growth rate
information held in the calcified structures called otoliths (fish ear
bones). These are Calcium carbonate concretions deposited in concentric
layers daily or annually.

My problem is that ideally i need a fixation method that I can leave my
field samples in indefinitely, but that will not react with or degrade the
otoliths. I used Bouins last time, but its acidic and degraded the otoliths.
Normally otoliths are stored in 95% ethanol when still embedded in tissue,
but the ethanol sometimes needs to be changed as it becomes acidic on
reaction with the tissues. Also this concentration of ethanol would
dehydrate the gonads and make them hard and not very good for histology.

The best thing I can think of is NBF, but I am not sure if this will react
with the otoliths, or how long I can leave the specimens in it. If I simply
fix and then transfer to 50 or 70 % ethanol (as would be normal), then I
stand to lose the otoliths (50 and 70 % ethanol is too acidic). What can I
buffer the Formalin with that is NOT a calcium chelator? And can I leave the
samples indefinitely in this until I get home and process them? All other
suggestions welcome.

Thanks
-- 
Dave Allsop
Ashworth Laboratories
Institute of Cell, Animal and Population Biology
University of Edinburgh                             X>   X>
Kings Buildings                             X>    X>
West Mains Road                                                 X>
EH3 9JT
Scotland

Email: David.Allsop@ed.ac.uk
Office Phone: (0131) 650 8667
Home Phone: (0131) 667 4423
WWW: http://homepage.mac.com/armydave/Frontpage.htm




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